The present invention relates to a lipidic vector system comprising a multi-layered lipid bilayer structure called a cochleate precipitate. The layers of the lipid bilayer structure are ionically bound together by a cation.
One or more therapeutic nucleotide sequences coding for a therapeutically beneficial molecule and one or more proteins that facilitate integration of the therapeutic nucleotide sequences physically associated with the cochleate precipitate.
The proteins are preferably adeno-associated virus (AAV) Rep 68 and Rep 78. The therapeutic nucleotide sequence is preferably positioned between AAV inverted terminal repeats (ITRs).
Upon contact with a lipid bilayer of a target cell, the cochleate vector structure delivers one or more of the therapeutic nucleotide sequences and one or more of the proteins to the interior of the target cell. Upon entry into the cell the one or more proteins facilitate the integration of the therapeutic nucleotide sequence(s) into the genome of the host cell.
Recent advances in molecular biology have increased the scientific understanding of the genetic basis for disease and have provided the tools for novel advances in gene therapy. For example, it is now possible to produce genetically engineered nucleotide sequences capable of expressing therapeutic molecules. Yet, major obstacles have remained, and one such obstacle has been the lack of effective means for delivering these therapeutic nucleotide sequences to the interior of a target cell in a form capable of integrating into the target cell""s genome. This invention relates to a vector delivery system capable of delivering genetic materials to the interior of a cell along with the molecules necessary for the integration of such genetic materials into the genome of the target cell.
The vector delivery system of the present invention comprises a lipidic structure called a cochleate precipitate or, simply, a cochleate. The cochleate comprises a multi-layered lipid bilayer structure. The multi-layered lipid bilayer structure generally comprises a membrane phospholipid containing a negatively charged head group and a cation such as calcium (Ca++). The cation serves as a bridge, ionically binding to the negatively-charged head groups of the phospholipid groups and thus linking together the individual lipid bilayers of the limit-layered structure.
Cochleates consist of alternating sheets of cation-complexed lipid. In a preferred mode of the invention, the multi-layered lipid bilayer structure exists as a continuous lipid bilayer sheet rolled up into a spiral conformation. The cation maintains the cochleate structure by ionically binding to the negatively charged head groups in the opposing lipid bilayers. For example, where the cation is C++, one positive charge of the Ca++ attracts a negatively charged phospholipid headgroup in one bilayer, and the other positive charge attracts a phospholipid headgroup in the opposing bilayer.
Cochleates are highly stable and can be stored in calcium-containing buffer. Cochleates can also be lyophilized to a powder, stored at room temperature, and reconstituted with liquid prior to administration.
While other lipidic vector delivery systems are known (See Lee et. al., xe2x80x9cLipidic Vector Systems for Gene Transferxe2x80x9d, Critical Reviews in Drug, Carrier Systems 14(2): 173-206 (1997)), they are typically in the form of liposomes and arc substantially different from the cochleate vector delivery system described herein. A liposome is a fluid-filled compartment bounded by a fluid lipid bilayer. Materials, such as DNA or protein, can be contained within a liposome, and such materials can be delivered to the interior of a cell by endocytic uptake or, in special instances, fusion of the liposome with the cell membrane.
Unlike cochleate structures, harsh environmental conditions, such as extreme pH levels or the presence of lipid degrading enzymes, render the lipid bilayer of a liposome susceptible to instability and compromise the membrane barrier. Compromise of the membrane barrier renders the contents of the liposome subject to attack by external elements. For example, degradative enzymes, such as proteases and nucleases can degrade proteins and polypeptides within the compromised liposome.
An additional difference between liposomes and cochleate is the presence of divalent cations. Cochleates are prepared by calcium induced fusion of liposomes. Cochleates can contain, for example, one-half the molar concentration of divalent cations relative to the molar concentration of phospholipids. The divalent cations organize the negatively charged lipid bilayers into solid sheets that roll or stack upon themselves, excluding water.
Cochleates are multi-layered, highly stable structures composed of non-toxic and non-inflammatory natural products. They are solid, lyophilizable precipitates containing little or no internal aqueous space. Whereas dehydration of liposomes, e.g., by lyophilization, destroys the morphology and integrity of liposomes, such dehydration has no adverse effects on cochleate morphology or functions. The layers of the cochleate are composed of alternating sheets of negatively charged phospholipid and calcium. This unique structure protects associated, xe2x80x9cencochleated,xe2x80x9d molecules from degradation. Since the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact even though the outer layers of the cochleate may be exposed to harsh environmental conditions or enzymes.
The formulation of integrative DNA protein complexes or the use of these complexes as gene transfer vehicles has not heretofore been described.
Calcium induced perturbations of membranes containing negatively charged lipids, and the subsequent, resulting membrane fusion events, are important mechanisms in many natural membrane fusion processes. Accordingly, while the definitive mechanism of cochleate delivery is unknown, it is hypothesized that cochleates act as membrane fusion intermediates. According to this theory, as the calcium rich membrane of a cochleate approaches a natural membrane, a perturbation and reordering of the cell membrane is induced, resulting in a fusion event between the outer layer of the cochleate and the cell membrane. This fusion results in the delivery of a small amount of the encochleated material into the cytoplasm of the cell. Theoretically, the cochleate can then break free of the cell and be available for another fusion event, either with this or another cell. Alternatively, the cochleate may be taken up by endocytosis, and fuse with the cellular membranes from within. In contrast, the lipid bilayer of most liposomes is highly thermodynamically stable and resists fusion with other liposomes or with other membrane bound structures.
The membrane fusion hypothesis is consistent with the observation that many naturally occurring membrane fusion events involve the interaction of calcium with negatively charged phospholipids, (generally phosphatidylserine and phosphatidylglycerol). This hypothesis is also consistent with experimental studies. For example, the ability of cochleates to mediate the induction of antigen specific, CD8+ cytotoxic lymphocytes supports the hypothesis that cochleates facilitate the cytoplasmic delivery of cochleate-associated macromolecules. And, immunological studies indicating a slow, long-term presentation of antigen are consistent with the theory that a single cochleate undergoes multiple fusion events over an extended period of time.
The present invention makes use of cochleates as delivery vehicles for genetic materials and proteins that facilitate integration of the genetic materials into the host genome. In one example, the genetic materials and proteins are from the adeno-associated virus (AAV). AAV is a naturally defective, single stranded DNA parvovirus that is commonly used as a vector.
Wild-type AAV generally requires co-infection with a helper virus in order to replicate. Without a helper virus, AAV integrates into the genome of the host cell and remains latent for extended periods of time. AAV has not been associated with any human disease, and the integration of AAV does not appear to affect cell replication. The propensity of the AAV genome to safely integrate into the genome of host cells makes it an attractive vector for gene therapy.
AAV encapsidated in viral structural proteins has been used as a vector for gene delivery. The present invention differs significantly in that it makes use of unencapsidated AAV genetic elements complexed with selected viral proteins, and packaged in cochleates.
The AAV genome is 4.68 kb in length and contains two open reading frames and two 145 bp inverted terminal repeats (ITRs) (Chatterjee et al, Science 258: 1485-88 (1992). The two open reading frames, located between the ITRs, contain the rep and cap genes, which contain proteins involved in replication and encapsidation, respectively. The AAV rep gene is transcribed from two promoters, p5 and P19. Transcription from the p5 promoter generates mRNA transcripts that encode the Rep 68 and 78 proteins. Rep 68 and 78 are known to mediate complex formation between AAV DNA and its integration site in human DNA (See Weitzman et al., xe2x80x9cAdeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA,xe2x80x9d Proc. Natl. Acad. Sci. USA 91:5808-5812 (1994)). These proteins bind specifically to the AAV terminal hairpin, formed by the terminal 125 bases, and possess helicase and site-specific endonuclease activities required for AAV replication. Id.
One approach to utilizing the integrative capacity of the AAV system is to co-transfect cells with a vector expressing the Rep proteins along with a vector containing a gene to be transfered flanked by the ITR""s. The difficulty with this approach is the potential chronic production of Rep and Cap proteins. Rep is known to have effects on the expression of some cellular genes and could be toxic at high levels. In particular, chronic expression of the proteins in hematopoietic stem cells could significantly alter their genetic program. This could lead to changes in differentiation patterns and potentials. The rep and cap genes can be excised and replaced by a nucleotide sequence containing one or more genes expressing one or more therapeutically effective molecules. However, as already mentioned, Rep 68 and Rep 78 are required for integration of the AAV genome into the genome of the target cell. The present invention solves this difficulty by packaging in vitro synthesized Rep 68 and Rep 78 proteins, along with AAV genetic material lacking the rep and cap genes. In a preferred embodiment of the invention, a cochleate is used to deliver an AAV DNA strand having the rep and cap genes excised and having one or more therapeutic genes spliced between the ITRs, copackaged with recombinant AAV Rep 68 and Rep 78 proteins.
A difficulty with the standard use of the AAV capsid to deliver AAV genetic materials is that the capsid size limits the length of the therapeutic nucleotide sequence that can be spliced between the ITRs. Due to the small size of the AAV capsid, nucleotide sequences over 5 kb integrate poorly. Since the AAV ITRs comprise at least 290 bases, approximately 4.7 kb are left for the therapeutic nucleotide sequence of interest. This upper limit rules out the use of larger nucleotide sequences that may be attractive candidates for gene therapy, such as the gene coding for dystrophin, the absence or dysfunction of which leads to Duchennic and Becker muscular dystrophy. In contrast, while the upper limit is not known for the number of bases that can be spliced between the AAV ITRs for delivery by the inventive cochleate vector system, it is predicted that this number will be much greater than the number of bases that can be spliced between the AAV ITRs for delivery by the AAV capsid. The capacity to deliver larger therapeutic nucleotide sequences should greatly increase the number of genetic disorders for which the AAV ITRs can be used as a vector.
Another advantage of the use of the cochleate as a packaging system for an AAV based plasmid is the ability to deliver the AAV genetic materials without contamination by helper viruses. The standard method for constructing AAV vectors involves the co-infection of a host cell with (1) the AAV genome having the rep and cap genes replaced by the gene of interest; (2) a helper plasmid containing the AAV rep and cap genes without the AAV ITRs, typically containing adenovirus promoters; and (3) a helper adenovirus. The AAV rep and cap genes produce the Rep and Cap proteins but cannot be encapsidated by the AAV capsid. The foreign gene flanked by the AAV ITRs is encapsidated in the AAV capsid by the action of the Rep and Cap proteins. The result is a mixture of recombinant AAV and adenovirus. The recombinant AAV must then be purified by buoyant density centrifugation with a risk of contamination by residual adenovirus. In contrast, the vector delivery systems of the present invention may be constructed without the aid of the helper adenovirus, thus eliminating the risk of adenovirus contamination.
An important aspect of the present invention is the transduction of hematopoietic stem cells. Hematopoietic stem cells are the undifferentiated, pluripotent progenitor cells from which other specialized blood cells develop. The ability to provide long-term correction of many genetic disorders of the hematopoietic cells is required for the reconstitution of other cells in the hematopoietic system.
Unfortunately, transduction of hematopoietic cells using, conventional retro-viral vectors requires the stimulation of such cells by cytokines. Cytokine stimulation is thought to destroy the pluripotent nature of these stem cells. The present inventors have surprisingly and unexpectedly discovered a means for transforming hematopoietic stem cells in the absence of cytokine stimulation, that results in the transduction of hematopoietic cells. Because this transduction occurs in the absence of cytokine stimulation, the integration into the host genome of the AAV genetic elements can be effected without disturbing the pluripotent capacity of hematopoietic stem cells. The present invention is therefore a major advance in the field of gene therapy, and particularly in the field of gene therapy for blood disorders.
It was not previously known that cochleates containing rep proteins and DNA with ITRs could serve as a vector for efficient delivery to the nucleus of the proteins and the DNA in a form suitable for rep protein catalyzed integration of the DNA.
It was also unexpected that the rep protein-DNA-cochleate complexes would retain functional association with long-term stability. Formulations stored for more than one year at 4xc2x0 C. exhibited no detectable loss in efficiency of gene transfer.